Antitoxins act to neutralize the effects of toxins on specific cells; production of a specific level of protective antitoxins or antibodies following immunization with toxoid vaccines containing a Diphtheria component is required for release of the vaccine lot. Currently, a second animal test is employed to evaluate the efficacy of the animal response to the injected vaccine. This research is focused on replacing this second animal test with an in-vitro cell test. This year, a collaborative study to determine the lower limits of detectable antibody in the cell and animal tests was unable to define the lower limit of the test due in part to variations in the cell test method. This has resulted in a reevaluation of the cell test method, incorporation freeze-dried horse antitoxin standards at 4, 2 and 1 unit levels, a uniform toxin dose, and a specific colorimetric determination of cell survival based in statistical variation of the test. Samples of unknowns and test reagents have been prepared to supply 5 collaborating labs with sufficient material to perform the new cell test as well as parallel in-vivo tests, with completion anticipated January 15, 1997.